1. Field of the Invention
The present invention relates generally to the fields of molecular genetics and veterinary medicine. More specifically, the present invention relates to the mutation of a DNA-dependent protein kinase protein which results in equine severe combined immunodeficiency and a diagnostic test to identify carriers of the mutation.
2. Description of the Related Art
V(D)J rearrangement is the molecular mechanism by which distinct gene segments (V, D, and J) are joined to form the coding sequences of immunoglobulin (Ig) and T cell receptor (TCR) variable regions. The rearrangement process is targeted by simple DNA sequence elements (recombination signal sequences, RSS) found immediately adjacent to all functional immune receptor gene segments and involves two double-stranded DNA cuts and subsequent re-ligations. This process results in the formation of two new DNA joints; coding joints which contain the coding information, and signal joints which contain the two recombination signal sequences. V(D)J rearrangement is mediated by a lymphoid-specific endonuclease (the RAG 1 and RAG 2 proteins) and ubiquitously expressed components of the double strand break repair pathway. The centrality of V(D)J recombination to the development of the vertebrate immune system is evident in situations where the process is defective.
Defective V(D)J recombination results in a complete block of B and T cell lymphopoiesis and the disease severe combined immunodeficiency (SCID). The first example of defective V(D)J recombination was described in 1983 by Bosma and colleagues, relating to a spontaneous mutation in mice that results in severe combined immunodeficiency (C.B-17 mice). In severe combined immunodeficiency mice, the only step in V(D)J recombination that appears to be impaired is resolution of coding ends. Instead of being resolved into functional immune receptors, cleaved coding ends accumulate abnormally in developing severe combined immunodeficiency lymphocytes. However, cleaved signal ends are resolved at a similar rate as in wild type lymphocytes in mice.
In 1990, it was demonstrated that the defect in severe combined immunodeficiency mice not only impairs V(D)J recombination, but also affects the more general process of double strand break repair (DSBR). This observation was the first to link V(D)J recombination and double strand break repair. In recent years it has been shown that at least four factors are required for both V(D)J recombination and double strand break repair: the Ku heterodimer, DNA-dependent protein kinase.sub.catalytic subunit (PK.sub.CS), XRCC4, and XRCC6.
Recently, defective DNA-dependent protein kinase.sub.catalytic subunit has been identified as the determinative factor in C.B-17 severe combined immunodeficiency mice. The DNA-end binding Ku heterodimer interacts with DNA-dependent protein kinase.sub.catalytic subunit to generate a protein kinase (DNA-PK) that is dependent on linear DNA for activation (i.e., DNA-dependent protein kinase). DNA-dependent protein kinase.sub.catalytic subunit is related to the phosphatidylinositol 3-kinase family whose members function in a variety of roles such as signal transduction by phosphorylation of phospholipids, control of cell cycle progression, and maintenance of telomere length.
Although DNA-dependent protein kinase.sub.catalytic subunit has been implicated in a variety of different processes, its precise role is unclear. The factor defective in the double strand break repair mutant CHO cell line XRI. In sum, defects in either the lymphocyte specific components of the V(D)J recombinase (RAG 1 -/- mice, RAG 2 -/- mice, RAG-deficient children) or any one of these double strand break repair factors (C.B-17 severe combined immunodeficiency mice, Arabian severe combined immunodeficiency foals, Ku80 -/- mice) results in B and T lymphocyte development being blocked and similar phenotypes are observed.
The occurrence of severe combined immunodeficiency in Arabian foals was initially reported in 1973 by McGuire and Poppie. Recently, it was demonstrated that severe combined immunodeficiency in Arabian foals is explained by a severe block in the generation of specific immune receptors because of defective V(D)J rearrangement. As is the case in murine severe combined immunodeficiency, equine severe combined immunodeficiency cells are hypersensitive to DNA damage because of severely diminished levels of DNA-dependent protein kinase.sub.catalytic subunit. However, these two genetic defects have important mechanistic differences. Unlike severe combined immunodeficiency mice that are preferentially defective in coding resolution, severe combined immunodeficiency foals are defective in both coding and signal resolution.
The prior art is deficient in the lack of effective means of determining the presence of the genetic deteminant for equine severe combined immunodeficiency in an animal of interest. The present invention fulfills this longstanding need and desire in the art.